CN110305894B - Rapid and efficient catalpa bungei genetic transformation method - Google Patents
Rapid and efficient catalpa bungei genetic transformation method Download PDFInfo
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Abstract
The invention discloses a rapid and efficient catalpa bungei genetic transformation method. The method uses the catalpa bungei cotyledon as an explant to induce embryonic callus, and then carries out agrobacterium-mediated genetic transformation, and mainly comprises the following steps: obtaining and proliferating genetic transformation receptors, including induction of cotyledon callus, induction and proliferation of embryogenic callus; preparing agrobacterium carrying target genes; genetic transformation of embryogenic callus; plant regeneration and rooting; detecting the regenerated plants; hardening and transplanting the transgenic plants. The mountain ash is genetically transformed by adopting the method, positive transformation plants are obtained in 80-100 days, and the transformation efficiency is 92.23% -97.14%. The method has the advantages of short period, simple operation and high genetic transformation efficiency, provides important technical support for the functional verification and molecular breeding of the catalpa bungei gene, and accelerates the cultivation process of new catalpa bungei varieties.
Description
Technical Field
The invention belongs to the field of a construction method of a genetic transformation system of plants, and relates to a rapid and efficient catalpa bungei genetic transformation method.
Background
Chinese ash (Chinese catalpa)Catalpa bungei) Is the catalpium (Bignoniaceae) genus (Catalpa Shop.) and has important effects in garden appreciation, ecological protection, urban greening and the like, has reputation of "wood king" since ancient times, and has very high scientific value and development and utilization value.
The catalpa bungei is extremely strong in adaptability and rapid in growth, is distributed in vast areas of the eastern seashore, west to Gansu, south to Yunnan and north to great wall of China, and is a precious and high-quality tree species which can be cultivated in a large area. However, in recent years, catalpa bungei pest and disease damage is serious, and the popularization process is limited. Therefore, the main problems to be solved urgently are to improve the disease and insect resistance of the catalpa bungei and to cultivate new varieties of high-quality disease and insect resistant catalpa bungei.
In a natural state, the catalpa bungei trees have incompatible selfing, low fruitlessness or fruiting rate, the breeding is mainly carried out by cuttage and grafting, the technical links are more, and the seedling period is long. Long-term asexual propagation results in the simplification of varieties, types and clones, and the shortage of catalpa bungei resources due to excessive development and utilization. In addition, the genetic improvement of catalpa bungei is mainly performed by crossbreeding, but the conventional breeding cycle is long and the variety improvement is limited. The method integrates target genes into the catalpa bungei genome by utilizing modern molecular biology and genetic engineering technology to obtain corresponding characters, is rapid, efficient and strong in purpose, greatly accelerates the breeding speed, improves the breeding efficiency, and is an effective means for solving the breeding of new varieties of the disease-resistant catalpa bungei. In recent years, research on tissue culture technology of catalpa bungei has been carried out, for example, yang Yan, which is published in "research on axillary bud proliferation and rapid propagation technology of catalpa bungei" from "forestry science and technology development"; han Chuangju et al, published in the academy of northwest Lin academy of academic, "research on tissue culture technique of catalpa bungei"; chinese patent CN104054580A "a tissue culture method for increasing the multiplication coefficient of catalpa bungei", and the like. However, few studies on the genetic transformation of catalpa bungei are reported at present, and only the Chinese patent CN107058374A "a construction method of catalpa bungei genetic transformation system" is invented, compared with the method, the method disclosed by the invention has the advantages that the high-efficiency transformation rate of 92.23% -97.14% is achieved without ultrasonic treatment for auxiliary infection transformation, the operation is simple, and the period is short.
Disclosure of Invention
The invention provides a rapid and efficient catalpa bungei genetic transformation method, which utilizes catalpa bungei cotyledons as explants to induce embryogenic callus to generate, further performs agrobacterium-mediated genetic transformation and plant regeneration, provides a mature technical approach for cultivating new varieties with disease and insect resistance by using a transgenic technology for catalpa bungei, and aims to accelerate the catalpa bungei molecular breeding process and produce transgenic plants.
The purpose of the invention is realized by the following technical scheme:
1. acquisition and propagation of genetically transformed receptors:
a) Induction of cotyledon callus: sterilizing mature catalpa bungei seeds by using 75% alcohol on the surface of 60 s, washing with sterile water for 2 times, soaking in 0.1% mercuric chloride solution for 18 min, washing with sterile water for 5 times, spreading on a callus induction culture medium, and carrying out dark culture at 25-28 ℃ for 20 d to obtain callus;
b) Induction of embryogenic callus: transferring the induced yellow-green cotyledon callus to an embryogenic callus induction culture medium, culturing at 25-28 ℃ under the light intensity of 1500lx and 12 h/d, and subculturing at intervals of 20 d until embryogenic callus is generated;
c) Propagation of embryogenic callus: transferring the induced embryonic callus to an embryonic callus proliferation culture medium, culturing at 25-28 ℃ under the light of 1500lx light intensity and 12 h/d, and subculturing every 20 d to proliferate the embryonic callus so as to meet the subsequent genetic transformation requirement;
2. preparation of Agrobacterium carrying the Gene of interest: introducing plant expression vector containing target gene into agrobacterium-mediated condition by freeze thawing method, picking out monoclonal,after the target gene PCR detection determines positive monoclonal, shake the bacteria overnight, collect the thalli, and then use the heavy suspension MS 0 (l) Resuspending;
3. genetic transformation of embryogenic callus: inoculating the proliferated embryogenic callus into the resuspended Agrobacterium liquid, completely immersing the embryogenic callus in the liquid at 100 rpm for 20-30 min, blotting the liquid on the surface of the embryogenic callus with absorbent paper, and spreading on co-culture medium MS 0 (s), culturing at 25-28 ℃ in dark for 24-48 h;
4. plant regeneration and rooting: transferring the embryogenic callus after co-culture to a differentiation culture medium added with antibiotics, stacking the embryogenic callus into a callus stack with proper size, carrying out selection culture on transformants, carrying out light culture at 25-28 ℃ under the light intensity of 1500lx and 12 h/d, subculturing to plant regeneration every 20 d, and transferring the regenerated seedling to a rooting culture medium for rooting culture, wherein the height of the regenerated seedling is 2.5-4 cm;
5. detecting the regenerated plants: carrying out expression detection of the reporter gene on the resistant regenerated plant seedlings in the step (4), simultaneously, taking leaves of the regenerated plants, extracting DNA, and carrying out PCR detection of target genes;
6. hardening and transplanting transgenic plants: and (3) unscrewing a bottle cap of the transgenic positive seedling with a strong root system, placing the transgenic positive seedling for 7d for hardening, taking out the positive seedling, cleaning a culture medium retained on the root, transferring the culture medium into a flowerpot filled with nutrient soil (peat soil: perlite = 3:2), placing the flowerpot in a heat-preservation shed, and removing the seedling after one week.
The culture media in the steps 1-4 are all adjusted to pH value of 5.8-6.0.
The callus induction medium in the step 1 a) comprises MS + 6-BA 0.5 mg/L + NAA 0.2 mg/L + sucrose 30 g/L + agar 3 g/L.
The embryogenic callus induction medium in the step 1 b) comprises DKW + 6-BA 0.6 mg/L + NAA 0.15 mg/L + sucrose 30 g/L + agar 3 g/L.
The component of the embryogenic callus proliferation medium in the step 1 c) is MS + 6-BA 0.6 mg/L + NAA 0.15 mg/L + sucrose 30 g/L + agar 3 g/L.
The resuspension solution in step 2 was MS 0 (l) MS + sucrose 30 g/L, resuspended agriculturalThe bacillus liquid concentration is OD600= 0.6-0.65.
The co-culture medium in step 3 is MS 0 (s), MS + sucrose 30 g/L + agar 3 g/L.
The differentiation medium added with antibiotics in the step 4 is DKW + 6-BA 0.6 mg/L + NAA 0.15 mg/L + ZT 0.2 mg/L + sucrose 30 g/L + 50-150 mg/L Kan + agar 3 g/L. The transformed embryogenic callus was transferred primarily to a differentiation medium containing 150 mg/L Kan, followed by 20 d subculture, transferred to a differentiation medium containing 100 mg/L Kan, and after 20 d transferred to a differentiation medium containing 50 mg/L Kan.
The callus with the diameter of 1-1.5 cm is the embryogenic callus pile with the proper size in the step 4.
The rooting medium in the step 5 is DKW + IBA 0.1 mg/L + NAA 0.1 mg/L + sucrose 20g/L + agar 3 g/L.
The invention provides at least the following beneficial effects:
1. the invention discloses a rapid and efficient catalpa bungei genetic transformation method. The invention provides a method for inducing and proliferating embryonic callus by taking catalpa bungei cotyledons as explants, and the obtained embryonic callus has strong differentiation and regeneration capacity and the differentiation rate reaches 100 percent. The gene is taken as a genetic transformation receptor, the number of cells of the transformation receptor is large, the differentiation and regeneration capacity of the transformed embryonic cells is strong, and somatic embryos generated by the transformed cells can be directly differentiated and developed into complete regeneration plants.
2. According to the method, the target gene is introduced into the plant genome by the method of soaking the embryogenic callus by the agrobacterium, auxiliary treatment such as ultrasonic wave, vacuum hydraulic pressure and the like is not needed, the operation is simple, the period is short, and the genetic transformation efficiency is high.
3. The transformed embryogenic callus is screened by a differentiation culture medium which is added with antibiotics and gradually decreased in 3 stages (150, 100, 50 mg/L Kan), so that the positive rate of a regenerated plant is greatly improved, and the period is shortened.
4. The invention takes woody plant (catalpa bungei) as a material, and compared with herbaceous plants, the establishment of a somatic cell embryo regeneration system of the woody plant is difficult and the genetic transformation efficiency is low. The invention takes the embryogenic callus with strong induced differentiation and regeneration capacity as the receptor to carry out the genetic transformation of the catalpa bungei, has simple operation, short period and high genetic transformation efficiency, provides important technical support for transgenic production and gene function verification, can be used for the molecular genetic improvement of the catalpa bungei, provides a new seed material for the innovation of catalpa bungei seed resources, and provides a test reference basis for the molecular breeding of other forest tree species.
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FIG. 1 shows the induction and proliferation of callus tissue She Peixing from catalpa bungei. A, embryogenic callus; b, the embryogenic callus was transferred to proliferation medium 10 d, 30 d for phenotype (C).
FIG. 2 shows genetic transformation of a recipient with a calli embryogenic mountain ash. A, preparing agrobacterium carrying a target gene; b, transferring the transformed embryogenic callus to a differentiation culture medium added with antibiotics for screening and differentiation culture; c, transgenic regeneration plants; and D, GUS staining analysis of regenerated plant seedlings.
FIG. 3 PCR detection of the target gene of transgenic regenerated plants. M, marker; p, plasmid positive control; 1-11, and carrying out PCR detection on target genes of randomly selected transgenic regeneration plants.
FIG. 4 hardening off and transplanting transgenic plants. A, hardening seedlings; b, transplanting; c, moving the glass tube into a heat preservation shed; d, hardening off the transgenic positive seedlings.
Detailed Description
The invention is further illustrated by the example of an infection with Agrobacterium EHA 105. It should be noted that the preparation of Agrobacterium-infected, transformation of Agrobacterium-infected plasmid, and detection of positive transformants are not different from previous methods.
(1) Acquisition and propagation of genetically transformed receptors
a) Induction of cotyledon callus: sterilizing mature catalpa bungei seeds with 75 percent alcohol on the surface by 60 s, washing with sterile water for 2 times, soaking in 0.1 percent mercuric chloride solution for 18 min, washing with sterile water for 5 times, paving on a callus induction culture medium, and carrying out dark culture on 20 d at 25-28 ℃ to obtain callus which is yellow green and compact;
b) Induction of embryogenic callus: transferring the induced yellow-green cotyledon callus to an embryogenic callus induction culture medium, culturing at 25-28 ℃ under the light of light intensity 1500lx and 12 h/d, subculturing once every 20 d, browning 10% of the callus, and generating embryogenic callus which is light yellow, compact and granular on the surface on the browned callus after subculturing for 2-3 times;
c) Propagation of embryogenic callus: transferring the induced embryogenic callus to an embryogenic callus proliferation culture medium, culturing at 25-28 ℃ under light with light intensity of 1500lx and 12 h/d, and subculturing once every 20 d to proliferate the embryogenic callus so as to meet the subsequent genetic transformation requirement.
(2) Preparation of Agrobacterium carrying Gene of interest
Introducing a plant expression vector pCambia2300 (Kan resistance and GUS reporter gene) into an agrobacterium-infected state by a freeze-thaw method, picking a monoclonal, shaking the bacteria overnight after determining a positive monoclonal by GUS gene PCR detection, collecting the bacteria, and resuspending a resuspension solution until OD600=0.6.
(3) Genetic transformation of embryogenic callus
And (3) inoculating the multiplied embryogenic callus into the resuspended agrobacterium liquid, completely immersing the embryogenic callus in the liquid, performing 100 rpm for 20 min, after absorbing the liquid on the surface of the embryogenic callus by absorbent paper, paving the embryogenic callus on a co-culture medium, performing dark culture at 25-28 ℃ for 48 h.
(4) Plant regeneration and rooting
Transferring the co-cultured embryogenic callus onto a differentiation culture medium containing 150 mg/L Kan, stacking the embryogenic callus into a callus stack with a proper size, and performing selective culture of a transformant at 25-28 ℃ under light with the light intensity of 1500lx and 12 h/d; 20 d, transferring the surviving resistant embryogenic callus to a differentiation culture medium containing 100 mg/L Kan, culturing 20 d, then transferring the surviving resistant embryogenic callus to a differentiation culture medium containing 50 mg/L Kan, and screening step by step, wherein the surviving resistant embryogenic callus and the regeneration plant can be subjected to subsequent GUS staining to identify the masculinity; and (3) obtaining regenerated seedlings after 2-3 months, shearing the regenerated seedlings after the height of the regenerated seedlings is 2.5-4 cm, inserting the stems of the sheared regenerated seedlings into a rooting culture medium, and performing rooting culture.
Table 1 different technical parameter settings in embodiments 2 to 7
Table 2 different technical parameter settings in embodiments 8-10
TABLE 3 different technical parameter settings in embodiments 11 to 16
The result shows that the transformation efficiency is increased and then reduced along with the increase of the concentration of the agrobacterium-infected bacterial liquid, and the optimal concentration is OD600=0.60-0.65; for the optimal infection time, the optimal infection time of different agrobacterium strain types is different, the EHA105 is 20 min, and the GV3101 is 30 min; the optimal dark incubation time was 48 h.
The regenerated plants obtained in embodiments 1 to 16 were subjected to the procedures (5) and (6) of the "a rapid and efficient method for genetic transformation of catalpa bungei" according to the present invention.
(5) Detecting the regenerated plants: and (4) performing GUS staining detection on the regenerated plant seedlings in the step (4), taking the leaves of the regenerated plants, extracting DNA, and performing PCR detection on GUS genes.
The specific primer sequences of the GUS gene are as follows: GUS forward sequence:5'-TGAATCCGCACCTCTGG-3', GUS reverse sequence:5'-TTCATTGTTTGCCTCCCT-3'.
The result shows that the PCR of the resistant regeneration plant sample obtains a specific fragment with the length of about 1100bp, the fragment is cut, recovered and sequenced, the sequencing result is compared with the GUS sequence, and the sequence consistency is 100 percent. 173 regenerated plants and 161 positive plants are detected in batches, and the positive rate reaches 92.53-97.14%. Therefore, through the layer-by-layer screening of each optimization step in the rapid and efficient catalpa bungei genetic transformation method, the largest transformed embryogenic callus stacks can be obtained in the shortest time, and transgenic positive plants with high genetic transformation efficiency can be efficiently obtained.
(6) Hardening and transplanting the transgenic plants: and (3) unscrewing a bottle cap of the transgenic positive seedling with a strong root system, placing the transgenic positive seedling for 7d for hardening, taking out the positive seedling, cleaning a culture medium retained on the root, transferring the culture medium into a flowerpot filled with nutrient soil (peat soil: perlite = 3:2), placing the flowerpot in a heat-preservation shed, and removing the seedling after one week.
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Claims (8)
1. A rapid and efficient catalpa bungei genetic transformation method is characterized by comprising the following steps:
(1) Acquisition and propagation of genetically transformed receptors:
a) Induction of cotyledon callus: sterilizing mature catalpa bungei seeds by using 75% alcohol on the surface of 60 s, washing with sterile water for 2 times, soaking in 0.1% mercuric chloride solution for 18 min, washing with sterile water for 5 times, spreading on a callus induction culture medium, and carrying out dark culture at 25-28 ℃ for 20 d to obtain callus;
b) Induction of embryogenic callus: transferring the induced yellow-green cotyledon callus to an embryogenic callus induction culture medium, culturing at 25-28 ℃ under the light intensity of 1500lx and 12 h/d, and subculturing at intervals of 20 d until embryogenic callus is generated;
c) Propagation of embryogenic callus: transferring the induced embryonic callus to an embryonic callus proliferation culture medium, culturing at 25-28 ℃ under the light of 1500lx light intensity and 12 h/d, and subculturing every 20 d to proliferate the embryonic callus so as to meet the subsequent genetic transformation requirement;
(2) Preparation of Agrobacterium carrying the Gene of interest: introducing a plant expression vector containing a target gene into an agrobacterium-mediated state by a freeze thawing method, picking a monoclonal, shaking the bacteria overnight after the target gene PCR detection determines a positive monoclonal, collecting the bacteria, and resuspending the bacteria by a resuspension solution; the agrobacterium is agrobacterium EHA105, and the concentration OD600 of the agrobacterium heavy suspension is =0.60-0.65; the resuspension is MS + sucrose 30 g/L;
(3) Genetic transformation of embryogenic callus: inoculating the proliferated embryogenic callus into the resuspended agrobacterium liquid, completely immersing the embryogenic callus in the liquid, 100 rpm for 20 min, sucking the liquid on the surface of the embryogenic callus with absorbent paper, and then flatly paving the liquid on a co-culture medium at 25-28 ℃ for dark culture of 48 h; the co-culture medium is MS + sucrose 30 g/L + agar 3 g/L;
(4) Plant regeneration and rooting: transferring the co-cultured embryogenic callus onto a differentiation culture medium containing 150 mg/L Kan, stacking the embryogenic callus into a callus stack with a proper size, and performing selective culture of a transformant at 25-28 ℃ under light with the light intensity of 1500lx and 12 h/d; 20 d, transferring the surviving resistant embryogenic callus to a differentiation culture medium containing 100 mg/L Kan, culturing 20 d, then transferring the surviving resistant embryogenic callus to a differentiation culture medium containing 50 mg/L Kan, and screening step by step; obtaining regenerated seedlings after 2-3 months, and transferring the regenerated seedlings with the height of 2.5-4 cm into a rooting culture medium for rooting culture;
(5) Detecting the regenerated plants: carrying out expression detection of a reporter gene on the resistant regenerated plant seedlings in the step (4), and simultaneously taking leaves of regenerated plants, extracting DNA (deoxyribonucleic acid) and carrying out PCR (polymerase chain reaction) detection of target genes;
(6) Hardening and transplanting transgenic plants: unscrewing a bottle cap of a transgenic positive seedling with a strong root system, placing for 7d, hardening, taking out the positive seedling, cleaning a culture medium retained on the root, moving into a flowerpot filled with nutrient soil, placing in a heat-insulating shed, and moving out after one week; the nutrient soil is formed by mixing peat soil and perlite in 3:2.
2. The rapid and efficient genetic transformation method for catalpa bungei as claimed in claim 1, which is characterized in that: all media were adjusted to pH 5.8-6.0.
3. The rapid and efficient genetic transformation method for catalpa bungei as claimed in claim 1, wherein: the callus induction medium is MS + 6-BA (6-Benzylaminopurine ) 0.5 mg/L + NAA (1-Naphthylacetic acid) 0.2 mg/L + sucrose 30 g/L + agar 3 g/L.
4. The rapid and efficient genetic transformation method for catalpa bungei as claimed in claim 1, wherein: the embryogenic callus induction medium was DKW + 6-BA 0.6 mg/L + NAA 0.15 mg/L + sucrose 30 g/L + agar 3 g/L.
5. The rapid and efficient genetic transformation method for catalpa bungei as claimed in claim 1, wherein: the embryogenic callus proliferation medium is MS + 6-BA 0.6 mg/L + NAA 0.15 mg/L + sucrose 30 g/L + agar 3 g/L.
6. The rapid and efficient genetic transformation method for catalpa bungei as claimed in claim 1, wherein: the differentiation medium added with antibiotics is DKW + 6-BA 0.6 mg/L + NAA 0.15 mg/L + ZT (Zeatin ) 0.2 mg/L + sucrose 30 g/L + 50-150 mg/L Kan (Kanamycin ) + agar 3 g/L; the transformed embryogenic callus was transferred primarily to a differentiation medium containing 150 mg/L Kan, followed by 20 d and transferred to a differentiation medium containing 100 mg/L Kan, followed by 20 d and transferred to a differentiation medium containing 50 mg/L Kan.
7. The rapid and efficient genetic transformation method for catalpa bungei as claimed in claim 1, which is characterized in that: the rooting medium is DKW + IBA (Indole-3-butyltric acid, indoleacetic acid) 0.1 mg/L + NAA 0.1 mg/L + sucrose 20g/L + agar 3 g/L.
8. The rapid and efficient genetic transformation method for catalpa bungei as claimed in claim 1, wherein: the diameter of the callus pile with proper size in the step (4) is 1-1.5 cm embryogenic callus pile.
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